CI-1033 restored expression of AHI 1 both at RNA and protein levels in AHI

lentivirus in K562 cells resulted in sharply increased CI-1033 HER2 inhibitor colony forming ability compared with control cells. Importantly, /sh4 cells reversed growth defi ciencies exhibited by suppression of AHI 1. To determine if in vitro eff ects could be replicated in vivo, NOD/SCID 2m / mice were injected subcutaneously with transduced cells to determine their ability to form tumors in vivo. Cells with suppression of AHI 1 failed or had signifi cantly reduced ability to form tumors compared with control K562 cells. In contrast, AHI 1 overexpressed cells rapidly generated larger tumors than the control cells within 3 wk. Strikingly, AHI/sh4 cells cotransduced with the AHI 1 construct could reverse in vivo growth defi ciencies resulting from knockdown of AHI 1.
These results demonstrate that Baicalein modulation of expression levels of AHI 1 in human CML cells regulates their transforming activity in vitro and in vivo. Suppression of AHI 1 expression in primitive BCR ABL transduced human CB cells and primary CML stem/ progenitor cells reduces their growth autonomy We next evaluated the eff ects of down regulation of AHI 1 in BCR ABL transduced primitive human CB stem/progenitor cells using lentiviral mediated RNA interference. Q RT PCR analysis indicated that endogenous AHI 1 expression was suppressed by 40 50% when the shRNA construct was introduced. Interestingly, BCR ABL transduced CB cells expanded rapidly in the presence or absence of GFs as expected, but the growth of the BCR ABL transduced cells with suppression of AHI 1 was signifi cantly suppressed.
Similarly, BCR ABL transduced CB cells with suppression of AHI 1 had up to 10 fold less CFC output compared with BCR ABL transduced CB cells. A similar reduction of CFC output was observed in the absence of GFs. Notably, burst forming units erythroids were especially increased in BCR ABL transduced CB cells compared with control cells, which remained predominantly myeloid as previously reported. Suppression of AHI 1 expression in BCR ABL transduced CB cells signifi cantly reduced myeloid CFC output, as well as primitive myeloid progenitors, suggesting that AHI 1 may play a role in regulation of overproduction of myeloid cells in CML.
To further elucidate the role of AHI 1 in primary CML stem/ progenitor cells, AHI 1 transcript levels were evaluated in pretreatment lin CD34 cells from 16 chronic phase patients with subsequent clinical responses to IM therapy and from 7 patients in blast crisis. Increased levels of AHI 1 expression were observed in lin CD34 CML stem/ progenitor cells from all patient samples studies, compared with lin CD34 normal BM cells. Interestingly, lin CD34 cells from IM nonresponders expressed higher levels of AHI 1 transcripts compared with the same cells isolated from IM responders. Lin CD34 cells from blast crisis patients also expressed relatively higher levels of AHI 1 transcripts. A lentiviral mediated AHI 1 shRNA construct was directly transduced into lin CD34 stem/progenitor cells isolated from three IM responders, three IM nonresponders, and three blast crisis patients. Lentiviral mediated suppression of AHI 1 expression resulted in a reduction in CFC output by 28 60% in lin CD34 CML cells from all patient samples stu