Fig 2: SPINK2 is highly expressed in CD34+ bone marrow cells(A) Uniform manifold approximation and projection (UMAP) graph shows the CD34+ cell clusters within human bone marrow as annotated by Setty et al.19(B) UMAP graph showing SPINK2 expression in CD34+ cells.(C) SPINK2 and SPINK9 expression values are reported as averaged normalized CPM (top graph), and percentage of SPINK2 or SPINK9 positive cells in different population of CD34+ cells within the bone marrow (bottom graph). A cell is considered positive if normalized CPM value is > 0. Numbers of analyzed cells (n) in each population are the following: HSC, hematopoietic stem cell (n = 4690); HMP, hematopoietic multipotent progenitor (n = 4306); CMP, common myeloid progenitor (n = 2328); GMP, granulocyte-monocyte progenitor (n = 3713); DP, dendritic progenitor (n = 2075); MP, megakaryocyte progenitor (n = 507); EP, erythroid progenitor (n = 3463); CLP, common lymphoid progenitor (n = 3237); (D) Averaged normalized CPM (left graph) and percentage of SPINK positive cells (right graph) in different populations of hematopoietic cells within the bone marrow of mouse C57BL/6. LT-HSC, Long Term-HSC (n = 216); HSCs/HMPs, hematopoietic stem and progenitor cells (n = 852); MP/EP/CMP/GMP, megakaryocyte progenitor/erythroid progenitor/common myeloid progenitor/granulocyte-monocyte progenitor (n = 851).

Fig 3: SPINK2 expression in human tissues and cancer cell lines(A) SPINK2 expression in 1,293 cancer cell lines assembled for tissue of origin in 26 groups. Averages of TPM±SD and percentage of positive cell lines (>10 TPM) are shown in top and bottom histograms, respectively.(B) SPINK2 expression levels in AML cell lines (n = 42); (C) in ALL cell lines (n = 45); and (D) in Lymphoma cell lines (n = 86).(E) qRT-PCR confirmed SPINK2 expression in Jurkat cell line, a model of ALL, while other leukemia cell lines (HL-60, K562, KASUMI-1) did not express SPINK2. HCT116, a colon cancer cell line, was used as calibrator for fold change calculation.

Fig 4: Schematic drawing illustrating the TI-D theory for SPINK2 role in hematopoietic stem cell niche

Fig 5: Kinetic properties of trypsin inhibition by SPINK2(A) Graph showing experimental initial reaction rates at different concentrations of SPINK2 (range 3.75–120 nM) and fixed concentration of total enzyme (0.0032 µM) and initial substrate (125 µM); Km was constrained at 2,450 µM. Ki was determined by fitting experimental data to Morrison’s equation by GraphPad Prism 8.0.2. Results are expressed as averages ±SEM of two independent experiments.(B) Initial reaction rates expressed as % of uninhibited reaction rate versus Log10 concentration of SPINK2. Blue lines and points are calculated values with a Ki = 0.011 µM; red points are experimental values.(C) Experimental “product vs. time” curves; enzymatic reactions were followed for 17 h by monitoring spectrophotometrically the conversion of the substrate FVR-NA in p-nitroaniline at 410 nm. Trypsin (3.2 nM) was incubated with two different SPINK2 concentrations (3.75 and 60 nM) corresponding to an inhibitor/enzyme ratio of 1.2 and 19, respectively.(D) Calculated data obtained with TI-Model, showing the different “product vs. time” curves obtained with temporary and persistent inhibition; (E) Graph showing the “% of initial SPINK2 concentration” vs. Time obtained by TI-Model with Ki = 3 nM and increasing concentration of SPINK2. A SPINK2kcat of 1.2 x 10-3 sec-1 and a fixed concentration of 3.2 nM trypsin were used in the simulation.(F) In vitro degradation of recombinant SPINK2 by trypsin at different times. Each incubation was performed in duplicate. Averages of densitometric areas of western blotting bands (shown in the inset) are expressed as percentages of value at time zero (t0).(G) Kinetic reaction scheme for enzyme activity, competitive enzyme inhibition, and inhibitor degradation. Differential rate equations and abbreviations for kinetic constants (k0-k5) and chemical species are also reported in the figure.